The conduction of electrical signals along myelinated nerve fibers depends on high-density clusters of ion channels at nodes of Ranvier. Demyelinating diseases or injuries disrupt ion channel clusters, block conduction of action potentials in axons, and lead to nervous system dysfunction. Neuron-glia interactions at paranodal junctions flanking nodes contribute to the formation and maintenance of nodes, and participate in bi-directional signaling between neurons and glia. The specific aims of this project are to determine the molecular mechanisms underlying formation and maintenance of 1) nodes of Ranvier and 2) paranodal junctions. A major impediment to these aims has been the relatively few proteins known to be at these sites. We identified a specialized paranodal cytoskeleton and will determine its role in organizing and maintaining neuroglial interactions and axon stability. We will ablate components of the paranodal cytoskeleton using in vivo electroporation to deliver shRNA plasmids. We speculate that a major cause of axon degeneration as seen in demyelinating diseases is disruption of the axonal cytoskeleton and altered neuroglial interactions. We will identify additional paranodal proteins using biochemical and proteomic methods. To determine the mechanisms underlying node of Ranvier formation, we will identify critical proteins, protein domains, protein-protein interactions, and nodal localization determinants. The proposed experiments are important for many nervous system diseases where neuron-glia interactions are disrupted (e.g., multiple sclerosis, Guillain-Barre syndrome, spinal cord injury). Identification of the mechanisms underlying neuron-glia interactions may contribute to better therapeutic treatments designed to preserve these interactions and their contribution to neuronal health and function.